Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31421
標題: 台灣茄科與十字花科作物Xanthomonas病原細菌之特性分析與檢測
Characterization and detection of pathogenic Xanthomonas species from solanaceous and cruciferous crops in Taiwan
作者: 呂昀陞
Lue, Yun-Sheng
關鍵字: 茄科植物細菌性斑點病菌;Xanthomonas euvesicatoria;細菌素;十字花科黑腐病菌;十字花科葉斑病菌;多引子聚合酶連鎖反應;抑制性減扣法;即時聚合酶連鎖反應;Xanthomonas gardneri;Xanthomonas perforans;Xanthomonas vesicatoria;Bacteriocin;Xanthomonas campestris pv. campestris;Xanthomonas campestris pv. raphani;multiplex PCR;Suppression subtractive hybridization, Real-timePCR
出版社: 植物病理學系所
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摘要: 
Xanthomonas屬植物病原細菌可引起包含糧食、蔬菜、果樹與花卉等作物之病害。十字花科作物與茄科作物為台灣重要之蔬菜作物,而危害此兩科作物之Xanthomonas屬植物病原細菌常藉由帶菌種子進行傳播,開發此兩類作物Xanthomonas屬植物病原細菌之檢測技術,則可防範Xanthomonas屬病原菌藉由種子進行傳播,此外由於台灣種子輸出入相當頻繁,防檢疫單位常須針對此些種傳病原細菌進行檢測,發展快速、靈敏、準確之檢測技術,可有效提升我國植物檢防疫技術能力。
本研究之目的係探討台灣地區引子茄科植物細菌性斑點病之Xanthomonas屬植物病原菌族群,並針對十字花科與茄科作物上之Xanthomonas屬植物病菌進行檢測技術之研發,本論文內容包含四章,第一章為前人研究,係針對近年來十字花科與茄科作物上Xanthomonas屬植物病原細菌相關文獻之回顧,並介紹基因體抑制性扣減法與即時聚合酶連鎖反應之原理與應用;第二章針對台灣地區引起茄科植物細菌性斑點病之Xanthomoas屬病原細菌進行分類探討,藉以釐清台灣茄科細菌性斑點病菌之族群,以作為病害防治之基礎資料;第三章以基因體抑制性扣減法針對十字花科作物中之Xanthomonas屬植物病原菌進行專一性核酸片段之篩選,再利用所獲得之專一性核酸片段進行檢測技術之開發,獲得可快速檢測十字花科黑腐病菌與葉斑病菌之PCR技術;第四章則是利用基因體抑制性扣減法針對茄科植物細菌性斑點病菌中之X. vesicatoria,進行專一性核酸片段之篩選,再利用所獲得之專一性核酸片段開發茄科植物細菌性斑點病菌X. vesicatoria之real-time PCR檢測技術,本研究所開發之相關檢測技術可供快速檢測十字花科與茄科作物Xanthomonas病原細菌之用。
本論文第二章題目為台灣Xanthomonas屬茄科植物細菌性斑點病菌之特性分析,其內容摘要如下:細菌性斑點病為茄科植物重要細菌性病害之一,Jones 等人將引起番茄與甜椒細菌性斑點病之Xanthomonas細菌重新分類為Xanthomonas euvesicatoria (Doidge) Jones、X. gardneri(Sutic) Jones、X. perforans Jones與X. vesicatoria(Doidge) Vauterin等四種。為了解台灣Xanthomonas屬茄科植物細菌性斑點病菌之特性與種類,本研究利用生化、PCR測試與碳素源利用等,分析台灣各地分離之53個茄科植物細菌性斑點病菌菌株。結果顯示其中40個菌株不具澱粉與果膠分解能力,而其餘13個菌株則同時具有澱粉與果膠分解能力;以X. axonopodis pv. vesicatoria (Doidge) Vauterin之專一性引子對RST 13/14與X. vesicatoria (Doidge) Vauterin 之專一性引子對C-2-2L/2R進行多引子聚合酵素連鎖反應 (multiplex PCR) 測試,其中40菌株可增幅出560 bp之X. euvesicatoria專一性DNA片段, 5個菌株可增幅出407 bp之X. vesicatoria專一性DNA片段,但有8個菌株無法以此兩組引子對增幅出預期之產物;利用Biolog 鑑定系統進行菌株碳素源利用測試,並以NT-SYS pc 2.0分析。測試結果顯示,供試之53個Xanthomonas菌株,有40株為X. euvesicatoria,8株為X. perforans及5株為X. vesicatoria。本研究測試之菌株中並未有X. gardneri特性之菌株。此外本研究鑑定為X. perforans之菌株,皆可產生對X. euvesicatoria菌株生長具抑制作用之細菌素,本篇為台灣X. perforans之首次報導。Xanthomonas perforans 在台灣之分佈及其生態特性值得進一步探討。
本論文第三章題目為應用多引子聚合酵素連鎖反應技術同時檢測十字花科黑腐病菌與細菌性葉斑病菌,其內容摘要如下:Xanthomonas campestris pv. campestris (Xcc)所引起之黑腐病為十字花科作物重要之病害,近年來X. campestris pv. raphani (Xcr)所引起之十字花科細菌性葉斑病已在許多國家發生,並造成嚴重之損失。感染或污染此兩種病原細菌之種子或種苗為其最初感染源,因此開發此兩種植物病原細菌之準確、靈敏且快速之檢測技術,實為病害診斷與植物檢疫所亟需。本研究應用基因體扣減法分別篩選出Xcc與Xcr特異性核酸片段,依其核酸序列設計出Xcc與Xcr之專一性引子對Xcc 2f/2r與Xcr 14f/14r,測試此兩組引子對之專一性與靈敏度,顯示此兩組引子對可分別對Xcc與Xcr增幅出200 bp與277 bp之核酸片段,其相對應之靈敏度分別為10 pg與100 pg。利用此兩組引子進行多引子聚合酶連鎖反應測試,顯示此技術可同時區別此兩種病原細菌之差異,並可應用此技術於十字花科黑腐病與細菌性葉斑病之診斷與種子帶菌之檢測。
本論文第四章題目為應用即時聚合酵素連鎖反應技術檢測茄科植物細菌性斑點病菌Xanthomonas vesicatoria,其內容摘要如下:細菌性斑點病為茄科植物重要細菌性病害之一,而X. vesicatoria更為歐盟所列管之重要植物檢疫病原細菌,本研究應用基因體扣減法技術篩選對X. vesicatoria具專一性之DNA片段進行設計,經以X. vesicatoria菌株Xv 92之基因體對X. euvesicatoria菌株Xv 62之基因體進行減扣,獲得對X. vesicatoria具專一性之選殖株Xv 92-6,經序列分析後,顯示該序列含有約400 bp之片段,並無相似序列可供比對,進一步以此序列進行引子對與核酸探針之設計,並將所設計之引子對Xv 1f/1r進行聚合酶連鎖反應 (PCR)測試,顯示引子對Xv 1f/1r可供專一性檢測X. vesicatoria之菌株,且其靈敏度可達50 pg,本研究為增加檢測X. vesicatoria之效率,同時亦設計可供進行TaqMan real-time PCR之引子對/探針組,將所設計之引子對Xvrt 1f/1r與Xvrt 2f/2r先以SYBR Green real-time PCR技術進行測試,顯示引子對Xvrt 2f/2r可對X. vesicatoria產生專一性訊號,因此利用此引子對與其所配合之核酸探針pXvrt 1進行TaqMan real-time PCR之測試,結果顯示所有供試之X. vesicatoria菌株,皆可在Ct值約為20時產生專一性訊號,且其靈敏度可達500 fg,檢測效率大於傳統PCR之效能約100倍,此外利用此技術進行人工帶菌種子之檢測,檢測之結果顯示,此技術可在Ct值小於30時,獲得X. vesicatoria之專一性訊號。本研究所開發出之TaqMan real-time PCR技術可供檢測X. vesicatoria種子帶菌之用。

The plant pathogenic Xanthomonas can infect several hosts including rice, fruit crop, horticulture crop and vegetable crop. The warm temperature and high humidity climate favor the diseases caused by Xanthomonas. Solanaceous and cruciferous plants are economically important vegetable crops in Taiwan. These xanthomonads associated with solanaceous and cruciferous plants often trigger serious losses. The contaminated seeds are the significant primary inoculum sources in the fields. Development of rapid and accurate detection techniques for these pathogens is critical for managing the diseases by establishing the system of disease-free seeds or transplants. The bacterial spot of tomato and sweet pepper caused by X. euvesicatoria (formerly name X. axonopodis pv. vesicatoria) and X. vesicatoria is an important disease in Taiwan. Strains of bacterial spot Xanthomonas (BSX) on tomato and pepper were reclassified into 4 species including X. euvesicatoria, X. garderni, X. perforans and X. vesicatoria by Jones et al. To understand the components of BSX in Taiwan, BSX strains collected from Taiwan were characterized by biochemical tests and PCR. The results revealed that the BSX strains tested can be characterized into X. euvesicatoria, X. perforans and X. vesicatoria, respectively. No X. garderni strain was found in this study. All strains of X. perforans characterized in this study produced the bacteriocin to inhibit the growth of X. euvesicatoria. In this study, the detection methods for pathogenic Xanthomonas species from solanaceous and cruciferous were developed using genomic suppress subtraction hybridization (SSH), respectively. The specific DNA fragments from X. campestris pv. campestris (Xcc) and X. c. pv. raphani (Xcr) were obtained by SSH and two specific primer pairs (Xcc 2f/2r and Xcr 14f/14r) were designed according to these specific DNA sequences. The primers of Xcc or Xcr can be used for detection and identification of Xcc or Xcr by multiplex PCR and the sensitivity of the two primer set were 10 pg and 100 pg , respectively. When the black rot and bacterial spot naturally infected-leaf tissues or seeds of crucifers were examined, Xcc and Xcr could be detected and identified specifically and simultaneously by the multiplex PCR. The detection technique developed in this study could be used to differentiate the diseases caused by Xcc and Xcr, and it could also be used to detect Xcc and Xcr from naturally infected crucifer seeds. The same strategy was used to obtain the specific DNA fragments from X. vesicatoria (Xv), and a conventional PCR based detection method was developed by the DNA sequences. The DNA sequence of Xv 92-6 was used to design primer pairs (Xv 1f/1r) for detection and identification of X. vesicatoria. A DNA fragment of 174 bp was specifically amplified from strains of X. vesicatoria by PCR with primer pair Xv 1f/1r. The detection sensitivity of primer pair Xv 1f/1r was 50 pg DNA. To increase the detection efficiency for detection of X. vesicatoria, primer pair Xvrt 2f/2r and TaqMan MGB probe pxvrt 1 was designed according tothe sequences of Xv 92-6. With this primer/probe set, strains of X. vesicatoria can be detected at the Ct values about 20. No specific signal was detected from the other tested bacteria when the Ct value at 40. The sensitivity of this method was 500 fg DNA. The primer pair Xvrt 2f/2r and TaqMan MGBprobe pXvrt1 developed in this study can be effectively used for identification and detection of X. vesicatoria. This is the first report of discovery X. perforans in Taiwan. And this is also the first report that the suppress subtraction hybridization was used for development of the PCR based detection techniques for Xanthomonas associated with solanaceous and cruciferous plants. These detection techniques can be effectively used in plant quarantine and seed industry.
URI: http://hdl.handle.net/11455/31421
其他識別: U0005-2008201011351600
Appears in Collections:植物病理學系

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